It is becoming apparent that myogenesis is controlled, at least in part, by proteins that bind to DNA regulatory elements in muscle-specific genes. This research will investigate a minimal, muscle-specific DNA regulatory element to understand how gene expression is controlled by complex protein DNA-interactions. A 28 bp fragment of the skeletal actin promoter is sufficient for muscle-specific expression when it is placed upstream of a nonmuscle TATA element. This muscle regulatory element (MRE) contains the core sequence motif, CC(A/T)6GG, which is referred to as CArG. The MRE is functionally different from nonmuscle elements, such as the c-fos SRE, yet they share the CC(A/T)6GG motif and bind to the same subset of nuclear factors. Presumably sequence differences between these elements alter the factor binding properties and this contributes to the different expression properties of the cognate promoters. The proteins that bind to the skeletal actin MRE are MAPF1 (and 2), SRF, and a recently identified factor, MF3. We have purified the MF3 binding activity to homogeneity from muscle. Molecular clones for MF3 and MAPF1 will be isolated. This will be accomplished by screening expression libraries with binding site probes or by screening with degenerate oligonucleotides designed from peptide sequence data. The analysis of these clones will provide clues about the nature of the heterogeneity found with the MAPF1 and 2 and the MF3 polypeptides. Transcript level measurements will indicate whether the factors are themselves regulated by muscle-specific promoters. Molecular clones will enable the production of recombinant factors and allow a genetic analysis of factor function. Purified factors will also be used to initiate studies on the assembly of the nucleoprotein complex. Experiments will be performed to evaluate whether these factors form higher-order complexes or compete for binding to the MRE. We are constructing a series of chimeric DNA elements to elucidate the molecular basis for the functional differences between the MRE and the SRE. Analyses of expression and factor binding properties of the synthetic elements will provide insights about the structural requirements for muscle-specific expression.